Regulation of stem cell gene production with specific and selective electric and electromagnetic fields

ABSTRACT

Methods and devices are described for the regulation of BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2, osteocalcin, and alkaline phosphatase mRNA in stem cells via capacitive coupling or inductive coupling of specific and selective electric and/or electromagnetic fields to the bone cells or other tissues containing the stem cells, where the specific and selective fields are generated by application of specific and selective signals to field generating devices disposed with respect to the stem cells so as to facilitate the treatment of diseased or injured bone and other tissues. The resulting methods and devices are useful for the targeted treatment of osteoporosis, osteopenia, osteonecrosis, fresh bone fractures, fractures at risk, nonunion, bone defects, spine fusion, and/or other conditions in which BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2, osteocalcin, and alkaline phosphatase mRNA and/or protein deficiencies in stem cells has been implicated.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present patent application is a Continuation-in-Part of U.S. patentapplication Ser. No. 13/242,606 filed Sep. 23, 2011, which is acontinuation of U.S. patent application Ser. No. 12/167,283 filed Jul.3, 2008 (now U.S. Pat. No. 8,065,015), which is a continuation of U.S.patent application Ser. No. 10/257,126 filed Oct. 8, 2002 (now U.S. Pat.No. 7,465,566), which is a U.S. National Phase of PCT/US01/05991 filedFeb. 23, 2001, which claims priority to U.S. Provisional Application No.60/184,491 filed Feb. 23, 2000. The contents of these applications arehereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention is directed to a method of up-regulating theexpression of those genes in stem cells that can be induced to regulatebone and cartilage growth and repair by application of an appropriateelectric or electromagnetic field. The genes include bone morphogeneticgenes (BMPs 2 and 4 in particular), transforming growth factor betagenes (TGF-beta 1, 2, and 3), fibroblastic growth factor-2 (FGF-2), andthe genes controlling alkaline phosphatase and osteocalcin expression inliving stem cells. The electric and electromagnetic fields generated bythe application of specific and selective electric and electromagneticsignals to coils, electrodes, or other field generating devices areapplied to injured or diseased bone, cartilage, and other tissues fortreatment.

BACKGROUND OF THE INVENTION

Although there has recently been much research on the interactions andactivity regarding the growth and repair of certain tissues and cells,there has been little to no research on the effect of electric and/orelectromagnetic fields on stem cells and the role that such stimulatedcells might have on the growth and repair of bone, cartilage, and othertissues. Such research may be useful in the development of newtreatments for a variety of medical problems.

The BMP family of genes include growth factors that are important in thepromotion of bone formation and maintenance, from proliferation ofpre-osteoblasts, to differentiation of pre-osteoblasts to matureosteoblasts, and to maintenance of the osteoblast throughout its lifespan. Transforming growth factors (TGF-beta 1, 2 and 3) control manycellular functions including growth, proliferation and differentiation.Fibroblastic growth factor (FGF-2) has been shown to have a positiveanabolic effect on bone formation in intact animals and to reduce boneloss in experimental models of osteoporosis. FGF-2 (also termed basicfibroblastic growth or bFGF) stimulates bone formation, decreasesosteoclast surfaces, and induces new bone spicules within the marrowcavity of bone.

Thus, up-regulation of the genes discussed above would be useful in thetreatment of the disease commonly known as osteoporosis, where bonedemineralizes and becomes abnormally rarefied. In osteoporosis, boneresorption exceeds bone formation, leading to bone weakening andpossible vertebral body fracture and collapse. While osteoporosis isgenerally thought to afflict the elderly, certain types of osteoporosismay affect persons of all ages whose bones are not subject to functionalstress. In such cases, patients may experience a significant loss ofcortical and cancellous bone during prolonged periods of immobilization.Elderly patients are known to experience bone loss due to disuse whenimmobilized after fracture of a bone, and such bone loss may ultimatelylead to a secondary fracture in an already osteoporotic skeleton.Diminished bone density may lead not only to vertebrae collapse, butalso to fractures of hips, lower arms, wrists, ankles as well asincapacitating pains. Alternative non-surgical therapies to induce boneformation in such diseases are needed.

Pulsed electromagnetic fields (PEMF) and capacitive coupling (CC) havebeen used widely to treat nonhealing fractures (nonunion) and relatedproblems in bone healing since approval by the Food and DrugAdministration in 1979. The original basis for the trial of this form oftherapy was the observation that physical stress on bone causes theappearance of tiny electric currents that, along with mechanical strain,were thought to be the mechanisms underlying transduction of thephysical stresses into a signal that promotes bone formation. Along withdirect electric field stimulation that was successful in the treatmentof nonunion, noninvasive technologies using PEMF and capacitive coupling(where the electrodes are placed on the skin in the treatment zone) werealso found to be effective. PEMFs generate small, induced currents(Faraday currents) in the highly-conductive extracellular fluid, whilecapacitive coupling directly causes currents in the tissues; both PEMFsand CC thereby mimic endogenous electrical currents.

The endogenous electrical currents, originally thought to be due tophenomena occurring at the surface of crystals in the bone, have beenshown to be due primarily to movement of fluid containing electrolytesin channels of the bone containing organic constituents with fixednegative charges, generating what are called “streaming potentials.”Studies of electrical phenomena in bone have demonstrated amechanical-electrical transduction mechanism that appears when bone ismechanically compressed, causing movement of fluid and electrolytes overthe surface of fixed negative charges on the surface of bone cells, thusproducing streaming potentials. These electrical potentials serve apurpose in bone, and, along with mechanical strain, lead to signaltransduction that is capable of stimulating bone cell synthesis of acalcifiable matrix, and, hence, the formation of bone.

It was reported in 1996 by the present inventor and others that a cyclicbiaxial 0.17% mechanical strain produces a significant increase inTGF-β₁ mRNA in cultured MC3T3-E1 bone cells in a Cooper dish (Brightonet al., Biochem. Biophys. Res. Commun. 229: 449-453, 1996). Severalsignificant studies followed in 1997. In one study it was reported thatthe same cyclic biaxial 0.17% mechanical strain produced a significantincrease in PDGF-A mRNA in similar bone cells (Brighton et al., Biochem.Biophys. Res. Commun. 43: 339-346, 1997). It was also reported that a 60kHz capacitively coupled electric field of 20 mV/cm produced asignificant increase in TGF-β₁ in similar bone cells in a Cooper dish(Brighton et al., Biochem. Biophys. Res. Commun. 237: 225-229, 1997). Ithas also been reported that chondrocyte matrix genes and proteins can beup-regulated by specific and selective electric fields (Wang, W., Wang,Z., Zhang, G., Clark, C. C., and Brighton, C. T., Clin. Orthp. andRelated Res., 427S: S163-173, 2004; Brighton, C. T., Wang, W., andClark, CC, Biochem. Biophys. Res. Commun., 342: 556-561, 2006). Further,it has been shown that the gene expression of bone morphogeneticproteins (BMPs) can also be up-regulated by specific and selectiveelectric fields that differ from the electric fields in various signalaspects from those signals that are specific and selective for articularcartilage (Wang, Z., Clark, C. C. and Brighton, C. T., J. Bone JointSurg., 88: 1053-1065, 2006).

In U.S. Pat. No. 7,465,566 to the present inventor, methods weredisclosed for determining the specific and selective electrical andelectromagnetic signals for use in creating fields for regulating targetgenes of diseased or injured tissues. The present invention builds uponthe techniques described therein by describing the method of regulatingexpression of the targeted genes in bone marrow stem cells. These genesbelong to the BMP super family, and include BMP 2 and 4, FGF-2, andTGF-beta 1, 2, 3, alkaline phosphatase, and osteocalcin. Throughapplication of a field generated by a specific and selective electricaland electromagnetic signal, the treatment of bone diseases and injuriesincluding osteoporosis, osteopenia, osteonecrosis, bone defects, freshfractures, fractures at risk, delayed union, nonunion, and as an adjunctin spinal fusion is described.

SUMMARY OF THE INVENTION

The present invention relates to regulating the gene expression of theBMP super family including, but not limited to, BMP 2 and 4, TGF beta 1,2, and 3, and fibroblastic growth factor-2 (FGF-2), alkalinephosphatase, and osteocalcin in stem cells (as an example) via theapplication of specific and selective electric and/or electromagneticfields generated by the application of specific and selective signals tocoils, electrodes, or other field generating devices adjacent the boneor tissue cells of interest. By performing sequential dose-responsecurves on the signal duration, amplitude, frequency, and duty cycle inwhich the effects of the resultant electric field are measured, theoptimal signal for up-regulating mRNA in stem cells was discovered. Theoptimal signal generated a capacitively coupled electric field with anamplitude of 20-40 mV/cm, a duration of 12 hours, a frequency of 60 kHz,and a duty cycle of 50-100%. In particular, the present inventionrelates to up-regulating the gene expression in stem cells via theapplication of fields generated by such signals.

In an exemplary embodiment of the invention, methods are provided tospecifically and selectively up-regulate the gene expression of stemcells mentioned above (as measured by mRNA) with capacitively coupledelectric fields, electromagnetic fields, or combined fields.Osteoporosis, osteopenia, osteonecrosis, fresh fractures, fractures atrisk, delayed unions, nonunion fractures, bone defects, as an adjunct inspinal fusion and the like are treated with a capacitively coupledelectric field of about 10-40 mV/cm with a field duration of about 12hours, a frequency of 60 kHz, a duty cycle of 50%-100%, and a sine waveconfiguration that causes the expression of BMP 2 and 4, FGF-2, and TGFbeta 1, 2, and 3 to be up-regulated. In accordance with the method ofthe invention, a “specific and selective” signal is a signal that haspredetermined characteristics of amplitude, duration, duty-cycle,frequency, and waveform that up-regulates the expression of the BMPsuper family of genes (specificity). This allows one to choose differentsignals to up-regulate the expressions of the genes in the BMP superfamily in order to achieve a given biological or therapeutic response(selectivity). The invention further relates to devices employing themethods described herein to generate specific and selective signals thatcreate electric and/or electromagnetic fields to up-regulate theexpression of the BMP super family of genes.

In related aspects, the invention relates to methods and devices for thetreatment of osteoporosis, osteopenia, osteonecrosis, fresh fractures,fractures at risk, delayed unions, nonunion fractures, bone defects, asan adjunct in spinal fusion and other therapies treating one or more ofthe above conditions. The method of the invention also includes themethodology for determining the “specific and selective” signal for BMP2 and 4, TGF beta-1, 2, and 3 and FGF-2 gene expression by methodicallyvarying the duration of a starting signal known to increase, orsuspected to increase, cellular production of the stated genes ofinterest. After finding the optimal duration, the amplitude of thesignal is varied for the optimal duration of time as determined by thegene expression of the above stated genes. The duty cycle, frequency,and waveform are varied methodically in the same dose response mannerwhile keeping the other signal characteristics constant. This process isrepeated until the optimal signal is determined that produces thegreatest increase in the expression of BMP 2 and 4, TGF beta-1, 2, and3, FGF-2, alkaline phosphatase, and osteocalcin. The optimal signal foreach of the stated genes may differ from one gene to another.

These and other aspects of the present invention will be elucidated inthe following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be apparent from the following detaileddescription of the invention taken in conjunction with the accompanyingdrawings, of which:

FIG. 1 is a scatter plot representation of the mRNA expression of BMPs 2and 4, TGF I, 2, and 3, FGF-2, alkaline phosphatase, and osteocalcinwhen stem cells are exposed to various capacitively coupled electricfield amplitudes. As indicated, the maximum expression for the variousmRNAs occurred within a range of signals from 10 mV/cm to 60 mV/cm.

FIG. 2 is a scatter plot representation of the mRNA expression of BMPs 2and 4, TGF I, 2, and 3, FGF-2, alkaline phosphatase, and osteocalcinwhen stem cells are exposed to a capacitively coupled electric fieldwith an amplitude of 20 mV/cm for various time durations. As indicated,the maximum expression for the various mRNAs occurred with a signalduration of 12 hours.

FIG. 3 is a scatter plot representation of the mRNA expression of BMPs 2and 4, TGF I, 2, and 3, FGF-2, alkaline phosphatase, and osteocalcinwhen stem cells are exposed to various capacitively coupled electricfield frequencies with a field amplitude of 20 mV/cm and a signalduration of 12 hours. As indicated, the maximum expression for thevarious mRNAs occurred with a frequency of 60 kHz.

FIG. 4 is a scatter plot representation of the mRNA expression of BMPs 2and 4, TGF I, 2, and 3, FGF-2, alkaline phosphatase, and osteocalcinwhen stem cells are exposed to various capacitively coupled electricfield duty cycles with a frequency of 60 kHz, a field amplitude of 20mV/cm, and a signal duration of 12 hours. As indicated, the maximumexpression for the various mRNAs occurred with a 50% to 100% duty cyclewith a sine wave configuration.

FIG. 5 is a diagram illustrating a device for the treatment ofosteoporosis of the spine, for example, in accordance with an exemplaryapplication of the present invention.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The invention will be described in detail below with reference to FIGS.1-5. Those skilled in the art will appreciate that the description givenherein with respect to those figures is for exemplary purposes only andis not intended in any way to limit the scope of the invention. Allquestions regarding the scope of the invention may be resolved byreferring to the appended claims.

The present invention is based on the discovery that the expression ofcertain stem cell genes can be regulated by the application of fieldsgenerated by specific and selective electric and/or electromagneticsignals applied to coils, electrodes, or other field generating devicesin the vicinity of the genes. In other words, it has been discovered bythe present inventor that there is a specific electric and/orelectromagnetic signal that generates a field for regulating genes instem cells in bone, cartilage and other tissues and that these specificsignals are capable of specifically and selectively regulating the genesin such cells. In particular, gene expression governing the growth,maintenance, repair, and degeneration or deterioration of tissues orcells can be regulated in accordance with the invention via theapplication of fields generated by application of specific and selectiveelectric and/or electromagnetic signals to field generating devices soas to produce a salutary clinical effect. Such discoveries are useful inthe development of treatment methods that target certain medicalconditions including osteoporosis, osteopenia, osteonecrosis, fresh bonefractures, fractures at risk, delayed union, nonunion, bone defects,spine fusion, and as an adjunct in the treatment of any one or more ofthe above conditions.

As used herein, the phrase “signal” is used to refer to a variety ofsignals including mechanical signals, ultrasound signals,electromagnetic signals and electric signals output by a device. It isto be understood that the term “field” as used herein refers to anelectrical field within targeted tissue, whether it is a combined fieldor a pulsed electromagnetic field or generated by direct current,capacitive coupling or inductive coupling.

The phrase “remote” is used to mean acting, acted on or controlled froma distance. “Remote” regulation refers to controlling the expression ofa gene from a distance. To provide “remotely” refers to providing from adistance. For example, providing a specific and selective signal from aremote source can refer to providing the signal from a source at adistance from a tissue or a cell, or from a source outside of orexternal to the body.

The phrase “specific and selective” signal means a signal that producesan electric field that has predetermined characteristics of amplitude,duration, duty cycle, frequency, and waveform that up-regulate ordown-regulate a targeted gene or targeted functionally complementarygenes (specificity). This allows one to choose different “specific andselective” signals to up-regulate or down-regulate expression of variousgenes in order to achieve a given biological or therapeutic response(selectivity).

The term “regulate” means to control gene expression. Regulate isunderstood to include both up-regulate and down-regulate. Up-regulatemeans to increase expression of a gene, while down-regulate means toinhibit or prevent expression of a gene.

“Functionally complementary” refers to two or more genes whoseexpressions are complementary or synergistic in a given cell or tissue.

“Tissue” refers to an aggregate of cells together with theirextracellular substances that form one of the structural materials of apatient. As used herein, the term “tissue” is intended to include anytissue of the body including muscle and organ tissue, tumor tissue, aswell as bone or cartilage tissue. Also, the term “tissue” as used hereinmay also refer to an individual cell.

“Patient” refers to an animal, preferably a mammal, more preferably ahuman.

The present invention provides treatment methods and devices that targetcertain tissues, cells or diseases. In particular, the gene expressionassociated with the repair process in injured or diseased tissues orcells can be regulated by the application of fields generated byelectric signals that are specific and selective for the genes to beregulated in the target tissues or cells. Gene expression can beup-regulated or down-regulated by the application of signals that arespecific and selective for each gene or each set of complementary genesso as to produce a beneficial clinical effect. For example, a particularspecific and selective signal may create an electric field thatup-regulates a certain desirable gene expression, while the same oranother particular specific and selective signal may create an electricfield that down-regulates a certain undesirable gene expression. Acertain gene may be up-regulated by a field generated by one particularspecific and selective signal and down-regulated by a field generated byanother specific and selective signal. Those skilled in the art willunderstand that certain diseased or injured tissues can be targeted fortreatment by regulating those genes governing the growth, maintenance,repair, and degeneration or deterioration of the tissues.

The methods and devices of the present invention are based onidentifying those signals that generate fields that are specific andselective for the gene expression associated with certain targeteddiseased or injured tissue. For example, fields of electricity appliedin various forms (e.g., capacitive coupling, inductive coupling, orcombined fields) can specifically and selectively regulate geneexpression in targeted tissues or cells in a patient's body by varyingthe frequency, amplitude, waveform or duty cycle of the signal appliedto the field generating device for generation of the applied field foreach selected gene or family of genes. The duration of time exposed toelectric field can also influence the capability of the electric fieldto specifically and selectively regulate gene expression in targetedtissues or cells in a patient's body. Specific and selective signals maygenerate electric fields for application to each gene systematicallyuntil the proper combination of frequency, amplitude, waveform, dutycycle, and duration is found that provides the desired effect on geneexpression.

It is to be understood that a variety of diseased or injured tissues ordisease states can be targeted for treatment because the specificity andselectivity of an electric field for a certain gene expression can beinfluenced by several factors. In particular, an electrical fieldgenerated from an electrical signal having the appropriate frequency,amplitude, waveform and/or duty cycle can be specific and selective forthe expression of certain genes and thus provide for targetedtreatments. Temporal factors (e.g., duration of time exposed to theelectrical field) can also influence the specificity and selectivity ofan electric field for a particular gene expression. The regulation ofgene expression may be more effective (or made possible) via theapplication of an electrical field for a particular duration of time.Therefore, those skilled in the art will understand that the presentinvention provides for varying the frequency, amplitude, waveform, dutycycle and/or duration of application of an electric signal thatgenerates an electric field until the electric field is found to bespecific and selective for certain gene expressions in order to providefor treatments targeting a variety of diseased or injured tissue ordiseases.

Thus, the present invention can provide for targeted treatments becauseit is possible to regulate expression of certain genes associated with aparticular diseased or injured tissue via the application of electricfields generated by specific and selective signals of appropriatefrequency, amplitude, waveform and/or duty cycle for an appropriateduration of time. The specificity and selectivity of a signal generatingan electrical field may thus be influenced so as to regulate theexpression of certain genes in order to target certain diseased orinjured tissue or disease states for treatment. In particular, thepresent invention provides for the targeted treatment of osteoporosis,osteopenia, osteonecrosis, fresh bone fractures, fractures at risk,nonunion, bone defects, spine fusion, and as an adjunct in the treatmentof one or any of the above conditions.

The devices of the present invention are capable of applying specificand selective signals to a field generating device for the generation ofa specific and selective field that is applied directly to diseased orinjured tissue and/or to the skin of a patient. The devices of thepresent invention may also provide for the remote application ofspecific and selective fields (e.g., application of a field at adistance from diseased or injured tissue yet which yields the desiredeffect within the targeted cells), although it will be appreciated thatcapacitively coupled devices must touch the subject's skin. The devicesof the present invention may include means for attaching the electrodesto the body of a patient in the vicinity of injured or diseased tissuein the case of capacitive coupling. For example, self-adherentconductive electrodes may be attached to the skin of the patient on bothsides of a fractured bone. As shown in FIG. 5, the device of the presentinvention may include self-adherent electrodes for attaching the deviceto the body of a patient. For example, the device of the presentinvention may include electrodes attached to a power unit that has aVELCRO® patch on the reverse side such that the power unit can beattached to a VELCRO® strap (not shown) fitted around a cast on thepatient. In the case of inductive coupling, the device of the presentinvention may include coils or other field generating device attached toa power unit in place of electrodes. The self-adherent strip electrodesalso may be attached to the back of a patient, with each electrode of apair of electrodes running parallel to the spine, with one electrode ofthe pair of electrodes placed longitudinally on one side of the spineand the other electrode of the pair placed longitudinally on theopposite side of the spine. In this case, the VELCRO® patch may be partof a garment worn by the patient.

The device of the present invention can be employed in a variety ofways. The device may be portable or may be temporarily or permanentlyattached to a patient's body. The device of the present invention ispreferably non-invasive. For example, the device of the presentinvention may be applied to the skin of a patient by application ofelectrodes adapted for contact with the skin of a patient for theapplication of electric fields generated by the predetermined specificand selective electric signals. Such signals may also be applied viacoils in which time varying currents flow, thus producing specific andselective electromagnetic fields that penetrate the tissue and createthe specific and selective electric fields in the target tissue. Forexample, the coils may be incorporated into the patient's clothing andplaced adjacent the patient's spine and/or hip as described in U.S. Pat.No. 7,158,835. The device of the present invention may also be capableof implantation in a patient, including implantation under the skin of apatient.

Those skilled in the art will further understand that the fieldgenerating devices of the present invention can be provided in a varietyof forms including a capacitively coupled power unit with programmed,multiple, switchable, specific and selective signals for application toone pair or to multiple pairs of electrodes, electromagnetic coils or asolenoid attached to a power unit with switchable, multiple, specificand selective signals, and an ultrasound stimulator with a power supplyfor generating specific and selective signals. Generally speaking, fieldgenerating device preference is based on patient acceptance and patientcompliance. The smallest and most portable unit available in the art atthe present time is a capacitive coupling unit; however, patients withextremely sensitive skin may prefer to use inductive coupling units. Onthe other hand, ultrasound units require the most patient cooperation,but may be desirable for use by certain patients.

EXAMPLE

The invention is demonstrated in the following example, which is forpurposes of illustration and is not intended to limit the scope of thepresent invention.

Materials and Methods

Human stem cells (5×10⁵ cells/cm²) were placed onto specially-modifiedCooper dishes. The cells were grown for ten days with the medium changedjust prior to beginning of the experimental condition. On day 10 theexperimental cell cultures throughout these studies were subjected to acapacitively coupled 60 kHz sine wave signal with an output of 44.81 Vpeak-to-peak. This produced a calculated-field strength in the culturemedium in the dishes of 20 mV/cm with a current density of 300 μA/cm².Control cell culture dishes were identical to those of the stimulateddishes except that the electrodes were not connected to a functiongenerator.

At the end of the experiment, total RNA was isolated using TRIzol,according to the manufacturer's instructions, and reversed transcription(RT) using SuperScript II reverse transcriptase was performed.Oligonucleotide primers to be used in the real time RT-PCR techniquewere selected from published cDNA sequences or designed using the PrimerExpress software program. Quantitative real-time analysis of RT-PCRproducts was performed using an ABI Prism® 7000 Sequence DetectionSystem.

The optimal signal for the desired up-regulation of the genes ofinterest, including BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2, alkalinephosphatase (ALP), and osteocalcin (BGP), was found systematically asfollows. An electrical signal known to cause creation of an electricfield that increases (or even just suspected to increase) cellularproduction of a given protein is taken as the starting signal fordetermining the specific signal for generating the electric field in thetargeted tissue for the gene expression (mRNA) of that protein. Adose-response curve is first performed by varying the amplitude of thesignal while holding all the other signal characteristics constant(duration, duty cycle, and frequency) (FIG. 1). This determines theoptimal amplitude of the starting signal for the gene expression of thatprotein. A second dose-response curve is then performed, this timevarying the duration of the electric field in the targeted tissue (inmV/cm) while holding the optimal amplitude and other signalcharacteristics constant (FIG. 2). A third dose response is performed,this time varying the signal frequency while holding constant theoptimal amplitude and optimal duration as found previously (FIG. 3). Afourth dose-response is performed varying the duty cycle from 100%(constant) to 10% or less while holding constant the optimal amplitude,duration, and frequency as found previously (FIG. 4). By this method, anoptimal signal is determined for producing the greatest increase in thegene expression of BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2, alkalinephosphatase, and osteocalcin.

FIG. 1 illustrates a scatter plot of human stem cell response after 10days of culture showing the effect of amplitude of stimulation on mRNAexpression of the eight genes of interest (BMP 2 and 4, TGF-beta 1, 2,and 3, FGF-2, alkaline phosphates, and osteocalcin). At 20 mV/cm thereis convergence of mRNA expression for seven of the genes that is notpresent at the other amplitude levels. It is further noted that theselected genes can give specific responses. For example, the optimalamplitude for TGF-beta 3 occurs at 10 mV/cm, BMP-4 at 60 mV/cm, etc. Thestem cells were stimulated for 12 hours at a frequency of 60 KHz at a50% duty cycle.

FIG. 2 illustrates a scatter plot of human stem cell response after 10days of culture showing the effect of duration of stimulation on mRNAexpression of the eight genes of interest (BMP 2 and 4, TGF-beta 1, 2,and 3, FGF-2, alkaline phosphates, and osteocalcin). The stem cells werestimulated for 2 to 24 hours with a signal frequency of 60 KHz, a dutycycle of 50%, and an amplitude of 20 mV/cm. It is noted that after 12hours of stimulation there was a convergence of the eight geneexpressions that is lacking at the other time periods tested. Also, asin FIG. 1, selected genes have an optimal response at specific hoursother than at 12 hours of stimulation.

FIG. 3 illustrates a scatter plot of human stem cell response after 10days of culture showing effect of frequency of stimulation on mRNAexpression of the eight genes of interest (BMP 2 and 4, TGF-beta 1, 2,and 3, FGF-2, alkaline phosphates, and osteocalcin). The stem cells werestimulated for the various signal frequencies at a duty cycle of 50%,and an amplitude of 20 mV/cm. It is noted that after 12 hours ofstimulation there were optimal expressions of the eight genes at afrequency of 60 kHz.

FIG. 4 illustrates a scatter plot of human stem cell response after 10days of culture showing the effect of the duty cycle on stimulation ofmRNA expression of the eight genes of interest (BMP 2 and 4, TGF-beta 1,2, and 3, FGF-2, alkaline phosphates, and osteocalcin). The duty cycleswere 12.5% (1 minute on/7 minutes off), 25% (1 minute on/3 minutes off),50% (1 minute on/1 minute off), 75% (1 minute on/20 seconds off), and100% (signal always on). It is noted that 50, 75, and 100% givevirtually identical results. A 50% duty cycle is chosen since itrequires less energy, and hence, gives longer battery life.

FIG. 5 illustrates a patient being treated for osteoporosis with acapacitive coupling device. The power pack (130) is worn in a clothpocket bag that is held at the waist with a cloth belt or can be slippedinto any pocket in the patient's clothing. Elements 110 a,b and 120 a,bindicate surface conductive cloth electrodes that are adherent to thepatient's back with conductive gel on either side of the lumbar spine atthe L1 to the L5 levels. The power unit delivers an optimized signal inaccordance with the invention, e.g., a 20 mV/cm sine wave signal at afrequency of 60 KHz, a duty cycle of 50%, and for a duration of 12 hoursper day. This is the optimal signal for stimulating the stem cellswithin the designated lumbar vertebrae for up-regulating mRNA expressionof the eight genes of interest (BMP 2 and 4, TGF-beta 1, 2, and 3,FGF-2, alkaline phosphates, and osteocalcin).

The present invention clearly shows that the optimal electric fielddescribed in the example (10 mV/cm-60 mV/cm, preferably 20 mV/cm, 12hours duration, 60 kHz frequency, and 50-100% duty cycle) cansignificantly up-regulate BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2,alkaline phosphatase, and osteocalcin and osteocalcin and, hence,increase bone formation in osteoporosis, osteopenia, osteonecrosis, bonefractures, fractures at risk, nonunion, bone defects, spine fusion, andas an adjunct in the treatment of one or any of the above. Also, FIGS.1-4 clearly show that there are differences among the responses of thevarious genes in each of the four dose-response studies. Those skilledin the art will appreciate that an appropriate electric field, asdescribed herein with capacitive coupling, is also equally effectivewith inductive coupling and all electromagnetic systems that produceequivalent, or nearly equivalent, electric field characteristics. Thoseskilled in the art will also appreciate that more unique signalcharacteristics may be discovered through more experimentation with moredata points (e.g., a 100±3% duty cycle for 30±3 min), but suchrelatively minor variations in each of the signal characteristics arebelieved to be within the level of those skilled in the art given theteachings herein.

Those skilled in the art will also appreciate that numerous othermodifications to the invention are possible within the scope of theinvention. For example, the optimal field described herein can beapplied to any bone via two or more appropriate surface electrodes, inpairs or strips, incorporated in braces, wraps, or casts, and deliveredby means of capacitive coupling. Also, the optimal field described herecan be applied to any bone via coil(s) or solenoid incorporated intobraces, wraps, or casts, and delivered by means of inductive coupling.Accordingly, the scope of the invention is not intended to be limited tothe preferred embodiment described above, but only by the appendedclaims.

1. A method of up-regulating the gene expression of BMP 2 and 4,TGF-beta 1, 2, and 3, FGF-2, and osteocalcin and alkaline phosphataseand of increasing bone formation by human stem cells in targeted tissue,comprising the steps of: generating at least one specific and selectivesignal having a frequency of approximately 60 kHz that when applied toone or more field generating devices operatively disposed with respectto said targeted tissue causes the generation of an electric and/orelectromagnetic field having an amplitude of approximately 10 to 60mV/cm in the targeted tissue that is specific and selective for theup-regulation of the gene expression of BMP 2 and 4, TGF-beta 1, 2, and3, FGF-2, and osteocalcin and alkaline phosphatase in the targetedtissue as measured by mRNA when said field is applied to the targetedtissue containing said BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2, andosteocalcin and alkaline phosphatase; and exposing said targeted tissueto the specific and selective field generated by said one or more fieldgenerating devices upon application of said at least one specific andselective signal thereto for a duration of approximately 12 hours per 24hour period at a duty cycle between 50% and 100% so as to selectivelyup-regulate the gene expression of BMP 2 and 4, TGF-beta 1, 2, and 3,FGF-2, osteocalcin, and alkaline phosphatase as measured by mRNA in saidtargeted tissue as a result of exposure to said field in the targetedtissue.
 2. The method of claim 1 wherein the generating step comprisesthe step of selectively varying the amplitude, duration, duty cycle,frequency, and waveform of the applied specific and selective signaluntil the gene expression of BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2,and osteocalcin and alkaline phosphatase as measured by mRNA in saidtargeted tissue as a result of exposure to the resultant specific andselective field in the targeted tissue is substantially increased. 3.The method of claim 1 wherein the generating step comprises the step ofgenerating an electric signal having a sine wave configuration where theresultant specific and selective field in the targeted tissue has anamplitude of approximately 20 mV/cm.
 4. The method of claim 1 whereinsaid generating step comprises the step of generating the specific andselective signal at a remote source and said exposing step comprises thestep of applying the specific and selective field to targeted stem celltissue.
 5. The method of claim 4 wherein the exposing step comprises thestep of applying the specific and selective field in the targeted tissuegenerated by the one or more field generating devices upon applicationof said at least one specific and selective signal thereto to thetargeted stem cell tissue through capacitive coupling or inductivecoupling.
 6. The method of claim 5 wherein the specific and selectivesignal applied to said electrodes causes the electrodes to generate acapacitive coupling electric field, and the specific and selectivesignal applied to said one or more coils causes said one or more coilsto generate an electromagnetic field or a combined field.
 7. A methodfor treating conditions including osteoporosis, osteopenia,osteonecrosis, bone defects, fresh fractures, fractures at risk, delayedunion, nonunion, and as an adjunct in spinal fusion, comprising:generating at least one specific and selective signal having a frequencyof approximately 60 kHz that when applied to one or more fieldgenerating devices operatively disposed with respect to targeted tissuecauses the generation of an electric and/or electromagnetic field havingan amplitude of approximately 10 to 60 mV/cm in stem cells in thetargeted tissue that is specific and selective for the up-regulation ofthe gene expression of BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2,osteocalcin, and alkaline phosphatase in the targeted tissue as measuredby mRNA when said field is applied to the targeted tissue containingsaid BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2, osteocalcin, and alkalinephosphatase; and exposing said targeted tissue to the specific andselective field generated by said one or more field generating devicesupon application of said at least one specific and selective signalthereto for a duration of approximately 12 hours per 24 hour period at aduty cycle between 50% and 100% so as to selectively up-regulate thegene expression of BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2,osteocalcin, and alkaline phosphatase as measured by mRNA in saidtargeted tissue as a result of exposure to said field in the targetedtissue.
 8. The method of claim 7 wherein the exposing step comprises thestep of applying either capacitively coupling or inductively couplingwith a specific and selective field to the targeted tissue.
 9. Themethod of claim 7 wherein the exposing step comprises the step ofapplying either an electromagnetic field or a combined field to thetargeted tissue.
 10. The method of claim 7 wherein the generating stepcomprises the step of generating an electric signal having a sine waveconfiguration where the resultant specific and selective field has anamplitude of approximately 20 mV/cm in the targeted tissue.
 11. Themethod of claim 7 wherein the generating step comprises the steps ofstarting with any electric signal that when applied to said one or morefield generating devices generates an electric and/or electromagneticfield that is known or thought to be effective on living cells,performing a first dose-response curve on the amplitude of stimulationof the field to determine an optimal amplitude; performing a seconddose-response curve on the optimal duration of the applied electricsignal using the optimal amplitude as previously found to determine anoptimal duration; performing a third dose-response curve on thefrequency of the applied electric signal keeping the optimal amplitudeand optimal duration as previously found to determine an optimalfrequency; performing a fourth dose-response curve varying the dutycycle of the applied electric signal and keeping the optimal amplitude,duration, and frequency as previously found to determine an optimal dutycycle, and keeping the optimal duration, amplitude, frequency and dutycycle constant while varying the wave form until an optimal wave formfor the up-regulation of the gene expression of BMP 2 and 4, TGF-beta 1,2, and 3, FGF-2, osteocalcin, and alkaline phosphatase as measured bymRNA and protein in the tissue is found.
 12. A device for the treatmentof osteoporosis, osteopenia, osteonecrosis, fresh bone fractures,fractures at risk, nonunion, bone defects, spine fusion, and as anadjunct in the treatment osteoporosis, osteopenia, osteonecrosis, freshbone fractures, fractures at risk, nonunion, bone defects, spine fusion,and/or other conditions in which BMP 2 and 4, TGF-beta 1, 2, and 3,FGF-2, osteocalcin, and alkaline phosphatase as measured by mRNA andprotein has been implicated in a patient, comprising: a signal sourcethat generates at least one specific and selective signal having afrequency of approximately 60 kHz, said signal source controlling andvarying duration of time of application of said at least one specificand selective signal for a duration of approximately 12 hours per 24hour period and said signal source varying a duty cycle of said specificand selective signal between 50% and 100%; and one or more fieldgenerating devices connected to the signal source so as to receive saidat least one specific and selective signal and that are operativelydisposed with respect to targeted tissue, said one or more fieldgenerating devices upon receipt of said at least one specific andselective signal causing the generation of a specific and selectiveelectric and/or electromagnetic field having an amplitude ofapproximately 10 to 60 mV/cm in the targeted tissue that is specific andselective for the up-regulation of the gene expressions of BMP 2 and 4,TGF-beta 1, 2, and 3, FGF-2, osteocalcin, and alkaline phosphataseand/or the up-regulation of the proteins in the targeted tissue asmeasured by mRNA when the field is applied to the targeted tissuecontaining BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2, osteocalcin, andalkaline phosphatase.
 13. The device of claim 12 further comprising aportable power unit that drives said signal source.
 14. The device ofclaim 12 further comprising means for attaching the one or more fieldgenerating devices to the body of a patient in the vicinity of targetedbone tissue.
 15. The device of claim 12 further comprising means forattaching the signal source to the body of a patient.
 16. The device ofclaim 12 wherein the field generated by application of said at least onespecific and selective signal to the one or more field generatingdevices is applied to said targeted tissue via capacitive coupling orinductive coupling.
 17. The device of claim 16 wherein the specific andselective signal has a sine wave configuration.
 18. A method ofdetermining a specific and selective signal that when applied to one ormore field generating device causes the generation of a field intargeted tissue that up-regulates BMP 2 and 4, TGF-beta 1, 2, and 3,FGF-2, osteocalcin, and alkaline phosphatase in the targeted tissue,comprising the steps of starting with a starting electric signal with asignal shape and frequency that when applied to said one or more fieldgenerating devices generates an electric and/or electromagnetic fieldthat is known or thought to affect cellular production of targetedtissue that up-regulates BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2,osteocalcin, and alkaline phosphatase mRNA, selectively varying aduration of application of said starting signal until a duration thatprovides a most significant increase in production of BMP 2 and 4,TGF-beta 1, 2, and 3, FGF-2, osteocalcin, and alkaline phosphatase mRNAis found, selectively varying an amplitude of the starting signal untilan amplitude that provides a most significant increase in production ofBMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2, osteocalcin, and alkalinephosphatase mRNA is found, selectively varying a duty cycle of thestarting signal until a duty cycle that provides a most significantincrease in production of BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2,osteocalcin, and alkaline phosphatase mRNA is found, and selectivelyvarying an on-off interval of the duty cycle of the signal until anon-off interval that provides a most significant increase in productionof BMP 2 and 4, TGF-beta 1, 2, and 3, FGF-2, osteocalcin, and alkalinephosphatase mRNA is found.
 19. A method as in claim 18, comprising thefurther steps of selectively varying a frequency and waveform of saidstarting signal, keeping other signal characteristics constant, until amost significant increase in production of BMP 2 and 4, TGF-beta 1, 2,and 3, FGF-2, osteocalcin, and alkaline phosphatase mRNA is found.